Several antiretroviral drugs are being tested as topical microbicides for the prevention of sexual HIV transmission. Because effective HIV prophylaxis would entail frequent use of topical antiretroviral drugs by healthy individuals, such use would need to be safe. However, in a recent comprehensive assessment of 1% tenofovir gel in vivo, conducted in the MTN-007 trial, we uncovered that it had profound effects on gene expression in the rectal mucosa, suggesting important changes in gut immune and self-repair homeostasis. When we followed up on these results using human primary vaginal epithelial cells, we found very similar changes. These changes, in particular pronounced impairment of mitochondrial function and the prospect of carcinogenicity, have the potential to contraindicate the use of tenofovir, and potentially of other drugs of the same DNA chain terminator class, as a topical microbicide. It is therefore imperative to extensively and immediately expand on these findings in order to corroborate tenofovir's side effects on the mucosa, to determine whether pericoital use is safer than daily use, and to compare its effects on the rectal and vaginal mucosa in vivo. In Aim 1 of this proposal we will address these issues using systems biology approaches in two trials testing 1% tenofovir gel, MTN-014 and MTN-017, using rectal and vaginal tissue specimens. Besides indirect suggestions in our data that tenofovir could increase neoplastic processes in the mucosa, a large body of data has demonstrated that DNA chain terminator drugs, like tenofovir and azidothymidine, are carcinogenic and mutagenic in a dose-dependent manner. Because of the potential gravity of this effect and the high concentrations of active drug achieved in mucosal tissues after topical application (>2 logs higher than achieved by oral dosing), we propose in Aim 2 to directly investigate the mucosal carcinogenicity of topical tenofovir and to determine whether non-DNA chain terminators like dapivirine might be safer alternatives. We propose innovative approaches designed to overcome the limitations of traditional rodent carcinogenicity models, incorporate human primary mucosal epithelial cells, and include an assessment of human papilloma virus (HPV) as a co-factor in tenofovir-induced carcinogenesis. These studies will help determine the relative suitability of drugs like tenofovir as topical prevention agents in comparison to non-DNA chain terminators like dapivirine, and provide novel tools for the assessment of the mucosal safety profile of candidate microbicide drugs, including the likelihood of carcinogenicity. Should our study confirm that DNA chain terminator drugs are too risky for mucosal application, it is hoped that we will find drugs like dapivirine to be effective and safer alternatives.